Method for the assessment of allergenicity

ABSTRACT

The invention relates to a method for the assessment of the allergenicity or the toxicity of a test compound and for the screening of at least two compounds simultaneously. The invention also relates to a method of characterizing a cell type for the present invention, as well as an assay kit for the high through-put screening of a compound. By contacting a cell culture of at least one cell type of animal origin with the test compound and measuring cytokine responses, the allergenicity of the test compound can be assessed.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of application Ser. No.09/689,554 filed Oct. 12, 2000 and claims, under 35 U.S.C. 119, priorityor the benefit of Danish application no. PA 1999 01486 filed Oct. 15,1999 and U.S. provisional application No. 60/160,543 filed Oct. 20,1999, the contents of which are fully incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The invention relates to a method for the assessment of theallergenicity or the toxicity of a test compound and for the screeningof at least two compounds simultaneously. The invention also relates isto a method of characterising a cell type for the present invention, aswell as an assay kit for the high through-put screening of a compound.

[0004] 2. Description of Related Art

[0005] Many compounds elicit allergic or toxic responses in animals aswell as in humans. In keeping with the ever increasing development ofnew compounds, and the resulting need of testing their potentialallergenicity or toxicity, methods for determining allergenicity andtoxicity have been developed. Historically, testing of compounds havebeen performed on animals, and to some extent humans, in lack ofavailable qualified technologies. For obvious reasons there is a publicand a professional demand and need to replace in vivo studies with invitro methods of testing compounds, such as allergenes or toxins.

[0006] Prior art attempts to uncover cell types, cellular elements andmolecules involved in an allergic response have aided the understandingof the complexity of this reaction chain. One such participating elementis cytokines. It is well-known that cytokines play a role as part of theestablishment of different immune responses.

[0007] Other participants are B and T lymphocytes. B and T cells are themediators of immunity, however their function is under the control ofantigen presenting cells, such as dendritic cells. Everson, M. P. et al.(Journal of Leukocyte Biology, vol. 59, 1996, pp.: 494-498) disclose howdifferent tissues induce the production of different T-cell cytokineprofiles. The study suggests that distinct dendritic cell populationsare responsible for the induction of T cell proliferation and cytokineproduction. Studies done by Secrist, H. et al. (J. Exp. Med, vol. 181,1995, pp.: 1081-1089) describe how the cytokine profile ofallergen-specific memory CD4+ T-cells can be modulated by the antigendose. They found that CD4+ T cells produced high levels of the cytokineinterleukin-4 (IL-4) when stimulated with low concentrations of allergenand low levels of IL-4 when stimulated with high concentrations ofallergen.

[0008] Driscoll, K. E. et al. (Environmental Health Perspectives, vol.105, 1997, pp.: 1159-1164) describe how inhaled particles, such asnoxious particles, elicit inflammation in the lung by effecting the lungepithelium cells. In vitro tests were performed on epithelial cells bystimulating with quartz, causing an increase in levels of the cytokineMIP-2 (macrophage inflammatory protein 2) measured as an increase inmRNA level. The investigators found that the response appeared to bedose related. Another research group have demonstrated in vitrosilica-induced mRNA expression of chemokines, such as MIP-2 in alveolarepithelial cells.

[0009] Prior art studies have mainly focused on the effect of a knownallergen on a particular cell population by observing the mechanisticproperties of the cells and the types of cytokines produced.

[0010] In patent application WO 99/07880 a method for the identificationof human allergens and T-lymphocyte antigens in vitro is disclosedwherein human naive T cells, macrophages/monocytes, immortalized B cellsand a test compound are mixed and it is determined whether the testcompound induces a response from the T-cells. One of the responsesmeasured is mentioned as being a cytokine response.

[0011] T cells recognize a processed antigen through interactions of a Tcell receptor, which is a result of clonal recombination, and through apeptide in an MHC molecule, which is individual dependent. This ofcourse represent an uncertainty when assessing whether a test compoundis an allergen since the response obtained is correlated to theindividual from which the cells originate and not to the test compoundper se. Thus, a need for a general test is still present.

SUMMARY OF THE INVENTION

[0012] The present invention relates to a method for the in vitroassessment of the allergenicity of a test compound, comprising the stepsof:

[0013] (a) obtaining a predetermined and precharacterized cell culturecomprising at least one cell type of animal, including human, origincapable of substantially non-specific interaction with the testcompound, and of responding with a cytokine expression upon interaction,

[0014] (b) contacting the cell culture with the test compound,

[0015] (c) defining a specific cytokine profile by determining for atleast one predetermined cytokine the cytokine response of the cellsexhibiting a substantially non-specific interaction with the testcompound, and

[0016] (d) correlating the cytokine profile to the allergenicity of thetest compound.

[0017] By the present invention it is possible to assess theallergenicity of a test compound in vitro by contacting the testcompound with a cell capable of eliciting a cytokine response anddetermine said response, based on the finding that a cytokine response,i.e. the type and level of cytokine secreted by the cell, variesdepending on the allergenicity of the test compound, and thencorrelating the IgE response to allergenicity studies carried out inanimals. Allergenicity has its ordinary meaning throughout the presentspecification, i.e. the ability of evoking an IgE response in animals,including humans.

[0018] The present invention further relates to a method wherein thecytokine response is determined in at least two cell cultures, eachcomprising one cell type, such as a co-culture.

[0019] In a further aspect of the invention the method as describedabove relates to the determination of membrane markers induced by thetest compound in cells exhibiting a substantially non-specificinteraction with the test compound.

[0020] Furthermore the invention describes a combined assay for thedetermination of the allergenicity and toxicity of a test compound.

[0021] Not only will the present invention bring benefit to animals andhumans, but it will also allow for the large scale screening ofpotential allergens and toxins, and thereby minimize operation expensesand time.

[0022] Another aspect of the present invention is a method for thesimultaneous screening of the allergenicity of at least two testcompounds, comprising the steps of:

[0023] (a) arranging a specific cell type in at least two separatecompartments of a cell culture as defined above,

[0024] (b) contacting the separated cell culture compartmentsindividually with a test compound,

[0025] (c) defining specific cytokine profiles by determining thecytokine responses of the respective cells exhibiting a substantiallynon-specific interaction with the test compound,

[0026] (d) correlating each cytokine profile to the allergenicity of thetest compounds.

[0027] In yet another aspect of the present invention a method fordetermining the toxicity of a test compound is provided, following thesteps as described above with respect to allergenicity.

[0028] Another aspect of the present invention is a method ofcharacterising a cell type as described above, comprising the steps of:

[0029] (a) obtaining a predetermined cell culture comprising one celltype of animal, including human, origin capable of substantiallynon-specific interaction with the test compound, and of responding witha cytokine expression upon interaction,

[0030] (b) identifying the cytokines that can be expressed by the cellculture upon non-specific interaction with the test compound by using anon-specific multivalent inducer,

[0031] (c) contacting at least a part of the cell culture with a testcompound, determining the level of the identified cytokines obtaining acytokine profile,

[0032] (d) immunizing an animal with the test compound and determiningthe resulting IgE level of the animal,

[0033] (e) correlating the cytokine profile to the IgE level determined,and

[0034] (f) repeating step c)-e) until at least a test compound of a highIgE level, a test compound of a low IgE level and a test compound of amedium IgE level have been tested.

[0035] A further aspect is an assay kit for high through-put screeningof a test compound, comprising,

[0036] (a) a cell culture comprising at least one animal, includinghuman, cell type,

[0037] (b) a cytokine determinant selected from at least one pair ofmonoclonal antibodies with specificity for a specific cytokine, at leastone cytokine-specific probe for mRNA detection, at least one set ofcytokine-specific primers for mRNA or cDNA detection,

[0038] (c) an assay device comprising at least two compartments.

[0039] Also, an aspect of the invention is the use of the assay forscreening of the allergenicity or toxicity of at least two testcompounds.

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] FIGS. 1A-D are four tables showing the cytokine responses whenepithelial cells are stimulated with lipo-polysaccharide (LPS) for apredetermined incubation time.

[0041]FIG. 2 shows the cytokine response for 3 different cytokines asreaction to the protease P modifications, wherein the protease P hasbeen modified with respect to allergenicity.

[0042] FIGS. 3A-D are graphs showing the reduction in IgE levels (a),the cytokine response of 3 different cytokines (b-d) in relation to thelength of PEG used to modify protease P.

[0043] FIGS. 4A-C are graphs showing the correlation between 3 differentcytokine levels and the IgE levels when stimulating with protease P.

[0044]FIG. 5 is a table showing the IgE levels and the cytokineresponses for 3 different cytokines when stimulating with a protease andLipolase.

DETAILED DESCRIPTION OF THE INVENTION

[0045] The present invention relates to an in vitro test for assessingthe allergenicity of a test compound. As described above theallergenicity of a test compound is normally assessed in animal studieswhereby the IgE level evoked by the test compound is indicative of theallergenicity of the test compound. However, in practice it is difficultto screen large amounts of test compounds for allergenicity. It is atedious and expensive task to test compounds through animal studies, andfor obvious ethical reasons it is desirable to minimize the use ofexperimental animals, which in turn is in agreement with the EUdirective on animal testing.

[0046] The present invention is based on the finding that the cytokineresponse of a cell capable of substantially non-specific interactionwith the test compound and of responding with a cytokine expression uponinteraction may be correlated to the IgE response that the same testcompound would elicit in animal studies.

[0047] The term “non-specific interaction” reflects the fact that someof the cells in the immune defence of the organism interactsnon-specifically with foreign substances, non-specifically as opposed tothe individual response that other cells, in particular T-cells areeliciting. The cells used according to the invention will elicit thecytokine response either by mere contact with the test compound or byuptake of the compound, such as by pinocytosis of the test compound.Accordingly, by interaction is meant the test compound is at least incontact with the cells and may be taken up by the cells. In the presentcontext the term “non-specific” means that a test compound will not bindto the cell surface and exert its effect through receptors specific forthe test compound. This is contrary to T cells which bind the processedantigen, such as an allergen through specific interactions by its T cellreceptor. Rather as mentioned earlier the uptake mechanisms by the cellsof the invention are not specific for the compounds in question.

[0048] The cells used according to the invention, capable of exhibitinga substantially non-specific interaction with the test compound, arepreferably epithelial cells irrespective of their location in theorganism. In common for epithelial cells are that they are part of theprimary defence of an organism against foreign substances.

[0049] In one embodiment of the invention the epithelial cells of theinvention are being derived from respiratory tract epithelial cells.Epithelial cells of the respiratory tract are directly exposed toairborne components of the exterior environment and a part of theprimary defence of the organism against foreign substances inhaled.Epithelial cells are often damaged in individuals suffering from asthmaand other allergic airway diseases.

[0050] In another embodiment of the invention the epithelial cells aregastro-intestinal tract epithelial cells.

[0051] In a further embodiment the at least one cell type iskeratinocytes. These cells are the major component of the epidermis,serving the role of protection of the underlying tissue.

[0052] Yet another at least one cell type of the invention for cellculturing is dendritic cells. The physiological role of dendritic cellsis to capture, process and present antigens, providing lymphocytes withco-stimulatory molecules, and to secrete the proper cytokines toinitiate immune responses. It has been found that dendritic cells arecapable of eliciting a cytokine response through a non-specificinteraction with a test compound.

[0053] Also, cells normally considered as part of the more individualand specific part of the immune system, such as macrophages, mast cells,and monocytes may be used according to the present invention, when theirability to elicit a non-specific cytokine response is used.

[0054] Macrophages defend the body against invading microorganisms byingestion via phagocytosis. Macrophages also serve as a scavenger forcleaning up damaged cells and cellular debris.

[0055] Yet another group of cells, endothelial cells, may be usedaccording to the invention.

[0056] Although the cell type used according to the invention may befrom any animal, it is preferred that the at least one cell type isderived from human tissue or a human blood cell.

[0057] Furthermore, it is preferred that the cell type used is relevantfor the main allergy location of the test compound assessed, i.e. testcompound suspected of causing pulmonary allergy are preferably tested ina system using respiratory epithelial cells.

[0058] Cytokines are a class of signalling molecules that help toregulate inflammatory processes and play an important role influencingthe response to antigens, including allergens. Cytokines contribute tothe recruitment of inflammatory an immune cells. They are secreted insmall amounts but are extremely potent, they act via receptors and arenot produced by unstimulated cells.

[0059] The production of cytokines by the cell types according to theinvention may be viewed as a primary response against a foreigncompound. Upon contact between the cell and the foreign compound thecell may bind the compound by unspecific mechanisms, such as unspecificreceptor binding. Following contact, the cells may internalize theforeign compound by pinocytosis or phagocytosis. The further enzymaticbreak down of the foreign compound intracellularly may trigger thesynthesis and secretion of cytokines. In the animal or human body thesecreted cytokines may act as messenger molecules effecting variousneighbouring cell types including immuno cells, such as T cells and Bcells, which in turn trigger a secondary response.

[0060] To date a wide variety of cytokines are known, however not allcytokines are produced by all cell types and furthermore, not allcytokines produced by the specific cell type may be secreted as aresponse to a non-specific interaction with a test compound.

[0061] Accordingly, the present invention further relates to a method ofcharacterising the specific cytokines of a predetermined cell type to bedetermined in relation to assessing the allergenicity of a testcompound. The method comprises the steps of

[0062] (a) obtaining a predetermined cell culture comprising one celltype of animal, including human, origin capable of substantiallynon-specific interaction with the test compound, and of responding witha cytokine expression upon interaction,

[0063] (b) identifying the cytokines that can be expressed by the cellculture upon non-specific interaction with the test compound by using anon-specific multivalent inducer,

[0064] (c) contacting a part of the cell culture with a test compound,determining the level of the identified cytokines obtaining a cytokineprofile,

[0065] (d) immunizing an animal with the test compound and determiningthe resulting IgE level of the animal,

[0066] (e) correlating the cytokine profile to the IgE level determined,and

[0067] (f) repeating step c)-e) until at least a test compound of a highIgE level, a test compound of a low IgE level and a test compound of amedium IgE level have been tested.

[0068] Once a cell type has been characterized as described above it ispossible to use a cell culture of the cell type in question forassessing the allergenicity of a test compound according to the presentinvention. Thus, by the term “precharacterized” is meant that thecytokine response from the specific cell type in relation to assessingallergenicity has been determined.

[0069] It is preferred that more than one cytokine is being assayed forin the method of assessing allergenicity of a test compound, such as atleast two cytokines are being assayed for. An improved prediction isobtained when at least four cytokines are being assayed for in themethod according to the invention.

[0070] In relation to human lung tissue epithelial cells the cytokinesassayed for are preferably Interleukin-6 (IL-6), Interleukin-8 (IL-8),MCP-1 and GM-colony stimulating factor (GM-CSF). For example, accordingto the invention a high allergenic test compound will elicit a low levelof the above mentioned cytokines assayed for, whereas a low allergenictest compound will elicit a high level of IL-8 and MCP-1 and a low levelof IL-6. For test compounds in between high and low allergenicity a highlevel of one or two cytokines is seen, whereas the level of the othercytokines being assayed for is low. Thereby it is possible to quantifythe allergenicity of a test compound based on the cytokine profileobtained by the present method.

[0071] The number and level of cytokines assessed are factorscorrelating to the allergenic potency of the test compound.

[0072] The cytokine profile may be quantified by a boolean method, i.e.+/−presence above a certain level, or by a direct numerical value of thelevel.

[0073] With respect to human lung tissue epithelial cells the booleanmethod can be exemplified as in Table 1: TABLE 1 Allergenicity/ CytokineIL-8 IL-6 MCP-1 High − − − Medium high + + − Medium low + + + Low + − +

[0074] In Table 1 + indicates the presence of the cytokine above acertain level, whereas − indicates the non-presence or presence underthe specified level.

[0075] It is an object of the present invention to provide for a methodwherein a cell culture comprises at least one cell type. However, it maybe an advantage to obtain a cytokine response from more than one celltype for each test compound assessed. By using more cell types it ispossible to obtain an even more improved method than when using one celltype, due to the various cytokines secreted by the different cell types.A method and an assay using more than one cell type may be arranged inseveral ways.

[0076] Two separate cell cultures comprising different cell types may becontacted with the same test compound and the cytokine profile may beobtained from each cell culture separately. The cytokine profiles maythen afterwards be combined and correlated to the allergenicity of thetest compound.

[0077] It is however preferred when using more than one cell culture,that the cultures are co-cultured. Thereby the natural environment forthe cells are mimiced to a greater degree, obtaining results having ahigher sensibility.

[0078] Accordingly, in an aspect of the invention two cell types areco-cultured. A co-culture according to the invention is a culturewherein the different cell types are sharing the same medium.

[0079] In a preferred embodiment of the invention the two cell types ofthe co-culture are physically separated. The cell types may be separatedby a semi-permeable membrane allowing for the passage of small moleculesfor instance the test compounds, and/or the test substance. The physicalset up of the co-culture may be such that one cell type is cultured inone compartment and the other cell type is cultured in a secondcompartment inserted into the first compartment, i.e. a sub compartmentof the first compartment.

[0080] The advantage of such a co-culture set-up may be that the secondcell type will be effected not only by the test compound but also by thecytokines produced by the cell type of the first cell culture inducing amore natural cytokine response of the second cell type.

[0081] In yet another embodiment the test compound may not be able topass through the membrane. This provides for an embodiment, wherein thecell culture in the second compartment is effected by the cytokinesproduced in the first cell culture, and not by the actual test compound.

[0082] In a further embodiment the test compound is added to a cultureof one cell type, upon which the supernatant is transferred to a secondculture of a second cell type. In this case the cytokines produced inthe first culture along with the test compound will effect the secondcell culture.

[0083] For all the above culture embodiments the test compound may beadded to the medium prior to the medium being added to the cellculture(s).

[0084] Preferred combinations of co-cultured cell types are epithelialcell types with non-epithelial cell types, such as a combinationswherein the first cell type is selected from: respiratory epithelialcells, gastrointestinal epithelial cells, keratinocytes, and the secondcell type is selected from dendritic cells, macrophages, mast cells,monocytes, and endothelial cells.

[0085] Accordingly, in one embodiment of the present invention the firstcell type is respiratory epithelial cells and the second cell type isselected from dendritic cells, macrophages, mast cells, monocytes, andendothelial cells.

[0086] In a preferred embodiment the first cell type is respiratoryepithelial cells and the second cell type is selected from dendriticcells or macrophages.

[0087] The cytokine response may be determined by any suitable method,such as by use of a determinant in the form of antibodies towards thecytokines produced or in the form of primers or probes for cDNA or mRNAencoding the cytokines produced.

[0088] Accordingly, the extracellular cytokine response may bedetermined by analysis with enzyme-linked immunosorbent assay (ELISA)using antibodies directed to the cytokines assayed for. Thereby aquantitative determination of the cytokines secreted from the cells asresponse to the interaction with the test compound is obtained.

[0089] In another embodiment the intracellular cytokine response ismeasured by use of ELISA as described above.

[0090] In yet another embodiment the cytokine response is determined byin-situ hybidization technique, preferably using probes directed to mRNAencoding the cytokines to be assayed for.

[0091] Also, the cytokine response may be determined by use ofpolymerase chain reaction (PCR) technique, in particular by usingprimers for mRNA encoding the cytokines to be assayed for.

[0092] The cytokines may be determined by analysis with the commerciallyavailable techniques of quantitative PCR technique or in-situ PCRtechnique.

[0093] The essential difference between in-situ PCR and quantitative PCRis the number of samples to be analysed. In order to give quantitativedata, in-situ PCR requires a number of identical cell cultures(representing the culture under investigation), each subjected to adifferent number of PCR cycles, in order to identify the dynamic rangeof the PCR for that specific cytokine/membrane marker in that specificcell culture.

[0094] Quantitative PCR requires one sample per cell culture. In suchsituation the development of the PCR product is followed over time, thusproviding quantitative data (ABI Prism 7700 Sequence Detection System).

[0095] In addition to the determination of the cytokine response fromthe cell type(s) used, it may be advantageous to determine membranemarkers induced by the test compound in cells exhibiting a substantiallynon-specific interaction with the test compound. In this respect it ispreferred to determine the membrane markers VCAM-1 or ICAM-1.

[0096] In one aspect of the invention the level and number of cytokinesassessed are factors correlating to the allergenic potency of the testcompound. For the purpose of the present invention the level and numberof cytokines assessed are factors correlating to low allergenic testcompounds, as well as the level and number of cytokines assessed arefactors correlating to high allergenic test compounds.

[0097] According to the invention a high allergenic test compound maynot elicit any cytokine response, i.e. when compared to the baselinecytokine level. In one embodiment of the present invention a highallergenic test compound may not elicit any cytokine response withrespect to the four cytokines tested in cell types described in theinvention. In a further embodiment a low allergenic test compound mayelicit a cytokine response of at least one cytokine, such as twodifferent cytokines. Yet in another embodiment of the invention a mediumhigh allergen may elicit a response of one cytokine, such as IL8.However, the absolute amount secreted may be far less than the responseof a low allergenic molecule as measured in picomole.

[0098] The method of assessing the allergenicity of a test compound isparticular useful in the screening of various test compounds withrespect to their allergenicity.

[0099] Accordingly, the present invention relates to a method for thesimultaneous screening of the allergenicity of at least two testcompounds, comprising the steps of:

[0100] (a) arranging a specific cell type in at least two separatecompartments of a cell culture as defined above,

[0101] (b) contacting the separated cell culture compartmentsindividually with a test compound,

[0102] (c) defining specific cytokine profiles by determining thecytokine response of the respective cells exhibiting a substantiallynon-specific interaction with the test compound,

[0103] (d) correlating each cytokine profile to the allergenicity of thetest compounds.

[0104] The screening method is of course preferably used for more than 2test compounds simultaneously, such as for about 10 test compounds, oreven about 100 test compounds simultaneously. The screening method isespecially suitable when testing compounds being modified with respectto their allergenicity.

[0105] Such modification of a test compound to affect its allergenicitycould be by mutation of a protein allergen in its IgE-specific epitopes.The location of these epitopes can be determined by several techniquessuch as those disclosed by WO 92/10755 (by U. Løvborg), by Walshet etal, J. Immunol. Methods, vol. 121, 1275-280, (1989), and by Schoofs etal. J. Immunol. vol. 140, 611-616, (1987). A preferred method foridentification of epitopes is by screening a random peptide library withantibodies (e.g. IgE antibodies) and the high binding peptide sequencesare aligned to identify a consensus sequence. These consensus sequences,in turn are compared with the sequence and 3D structure of a parentprotein, which is desired to mutate for reduction of allergenicity, inorder to identify the linear and structural epitopes of the parentprotein.

[0106] When searching for such a protein variant with improvedproperties, it may be an advantage to establish a library of diversifiedmutants each having one or more changed amino acids introduced andselecting those variants which show improved properties. This desirableproperty could be reduced allergenicity expressed by a favorablecytokine response in the methods of this invention.

[0107] A diversified library can be established by a range of techniquesknown to the person skilled in the art (Reetz M T; Jaeger K E, inBiocatalysis—from Discovery to Application edited by Fessner W D, Vol.200, pp. 31-57 (1999); Stemmer, Nature, vol. 370, p.389-391, (1994);Zhao and Arnold, Proc. Natl. Acad. Sci., USA, vol. 94, pp. 7997-8000,(1997); or Yano et al., Proc. Natl. Acad. Sci., USA, vol. 95, pp5511-5515, (1998); and Deng S J, et al. Proc. Natl. Acad. Sci., USA,Vol. 92(11), 4992-4996 (1995)).

[0108] These include, but are not limited to, ‘spiked mutagenesis’, inwhich certain positions of the protein sequence are randomized bycarring out PCR mutagenesis using one or more oligonucleotide primerswhich are synthesized using a mixture of nucleotides for certainpositions (Lanio T, Jeltsch A, Biotechniques, Vol. 25(6),958,962,964-965 (1998)). The mixtures of oligonucleotides used withineach triplet can be designed such that the corresponding amino acid ofthe mutated gene product is randomized within some predetermineddistribution function. Algorithms exist, which facilitate this design(Jensen L J et al., Nucleic Acids Research, Vol. 26(3), 697-702 (1998)).

[0109] Another method of creating a diversified gene library is by using‘family shuffling’ using a number of different, but homologous, genes asa starting point (Stemmer, Nature, vol. 370, p.389-391, 1994). Thesegenes are fragmented and the fragments used as templates for a PCRreaction, which generates hybrid gene products incoporating sequenceelements from several of the parent genes.

[0110] As described in the above mentioned references, these approachesmay be used in parallel or in series to create directed evolution of aprotein backbone to acquire desired properties, such as lowallergenicity.

[0111] In a preferred embodiment, substitutions are found by a methodcomprising the following steps: 1) a range of substitutions, additions,and/or deletions are listed encompassing several epitope areas, 2) alibrary is designed which introduces a randomized subset of thesechanges in the amino acid sequence into the target gene, e.g. by randommutagenesis, 3) the library is expressed, and preferred variants areselected using the methods of the present invention.

[0112] In a more preferred embodiment, the preferred variants areselected in an automated assay system, capable of processing manyvariants of the diversified library in a high throughput format. In thatcase, the test compound is typically a protein secreted from cells thatcan be cultured in a titer-plate format (e.g. bacterial or yeast cellsor others). The cell supernatant will, in addition to the test compound,comprise a number of other compounds which may cause a cytokine responsedifferent from the baseline. These could be intact cells; cell wall orother organelle fragments from lysed cells; lipopolysaccharides;glycoproteins; small molecules etc. It may be an advantage to preventthese compounds from contacting the cells of the current invention inorder to minimize background signaling.

[0113] In an even more preferred embodiment, at least two assays arecarried out in parallel: one being an assay using the methods of thisinvention, and the other being a functional assay for the test compound.In the case of a protease test compound, the functional assay may be aprotease activity assay. A protease activity assay may be determinedusing the substrate Suc-Ala-Ala-Pro-Phe-p-nitroaniline. The proteasecleaves the bond between the peptide and p-nitroaniline to give avisible yellow colour absorbing at 405 nm. Thus, the substrate and aprotease solution are mixed and the absorbance is monitored at 405 nm asa function of time as a measure of the protease activity in the sample.The scope of these embodiments of the invention is by no means limitedto protease, which serves only to provide an example.

[0114] In a most preferred embodiment, this method is supplemented withadditional rounds of screening and/or family shuffling of hits from thefirst round of screening (J. E. Ness, et al, Nature Biotechnology, vol.17, pp. 893-896, (1999)) and/or combination with other methods ofreducing allergenicity by genetic means (such as that disclosed in Wo92/10755).

[0115] The test compound may be any compound suspected of eliciting anallergenic response in animals, including humans. The allergenicresponse may be any allergenic response, such as a pulmonary allergenicresponse caused by inhaled compounds, or a skin allergy caused by skincontact with the allergen, or even gastrointestinal allergy caused bydigested allergens.

[0116] Accordingly, the test compounds may be any protein, such as aglycoprotein, or a lipoprotein, or a proteolipid, or a phospholipid. Theterm protein is intended to also include peptides and polypeptides.

[0117] In particular the test compound may be an enzyme or an enzymevariant, such as glycosyl hydrolases, carbohydrases, peroxidases,proteases, lipases, phytases, polysaccharide lyases, oxidoreductases,transglutaminases and glycose-isomerases, in particular the following:

[0118] Proteases

[0119] Proteases (i.e. enzymes classified under the EnzymeClassification number E.C. 3.4 in accordance with the Recommendations(1992) of the International Union of Biochemistry and Molecular Biology(IUBMB)) include proteases within this group.

[0120] Examples include proteases selected from those classified underthe Enzyme Classification (E.C.) numbers:

[0121] 3.4.11 (i.e. so-called aminopeptidases), including 3.4.11.5(Prolyl aminopeptidase), 3.4.11.9 (X-pro aminopeptidase), 3.4.11.10(Bacterial leucyl aminopeptidase), 3.4.11.12 (Thermophilicaminopeptidase), 3.4.11.15 (Lysyl aminopeptidase), 3.4.11.17(Tryptophanyl aminopeptidase), 3.4.11.18 (Methionyl aminopeptidase);

[0122] 3.4.21 (i.e. so-called serine endopeptidases), including 3.4.21.1(Chymotrypsin), 3.4.21.4 (Trypsin), 3.4.21.25 (Cucumisin), 3.4.21.32(Brachyurin), 3.4.21.48 (Cerevisin) and 3.4.21.62 (Subtilisin);

[0123] 3.4.22 (i.e. so-called cysteine endopeptidases), including3.4.22.2 (Papain), 3.4.22.3 (Ficain), 3.4.22.6 (Chymopapain), 3.4.22.7(Asclepain), 3.4.22.14 (Actinidain), 3.4.22.30 (Caricain) and 3.4.22.31(Ananain);

[0124] 3.4.23 (i.e. so-called aspartic endopeptidases), including3.4.23.1 (Pepsin A), 3.4.23.18 (Aspergillopepsin I), 3.4.23.20(Penicillopepsin) and 3.4.23.25 (Saccharopepsin); and

[0125] 3.4.24 (i.e. so-called metalloendopeptidases), including3.4.24.28 (Bacillolysin).

[0126] Examples of relevant subtilisins comprise subtilisin BPN′,subtilisin amylosacchariticus, subtilisin 168, subtilisinmesentericopeptidase, subtilisin Carlsberg, subtilisin DY, subtilisin309, subtilisin 147, thermitase, aqualysin, Bacillus PB92 protease,proteinase K, Protease TW7, and Protease TW3, and Bacillus PD498 (WO93/24623).

[0127] Specific examples of such readily available commercial proteasesinclude Esperase®, Alcalase®, Neutrase®, Dyrazym®, Savinase®, Pyrase®,Pancreatic Trypsin NOVO (PTN), Bio-Feed™ Pro, Clear-Lens Pro (allenzymes available from Novo Nordisk A/S).

[0128] Examples of other commercial proteases include Maxtase®,Maxacal®, Maxapem® marketed by Gist-Brocades N. V., Opticlean® marketedby Solvay et Cie. and Purafect® marketed by Genencor International.

[0129] Examples of protease variants are disclosed in EP 130.756(Genentech), EP 214.435 (Henkel), WO 87/04461 (Amgen), WO 87/05050(Genex), EP 251.446 (Genencor), EP 260.105 (Genencor), Thomas et al.,(1985), Nature. 318, p. 375-376, Thomas et al., (1987), J. Mol. Biol.,193, pp. 803-813, Russel et al., (1987), Nature, 328, p. 496-500, WO88/08028 (Genex), WO 88/08033 (Amgen), WO 89/06279 (Novo Nordisk A/S),WO 91/00345 (Novo Nordisk A/S), EP 525 610 (Solvay) and WO 94/02618(Gist-Brocades N. V.).

[0130] The activity of proteases and variants thereof can be determinedas described in “Methods of Enzymatic Analysis”, third edition, 1984,Verlag Chemie, Weinheim, vol. 5.

[0131] Lipases

[0132] Lipases (i.e. enzymes classified under the Enzyme Classificationnumber E.C. 3.1.1 (Carboxylic Ester Hydrolases) in accordance with theRecommendations (1992) of the International Union of Biochemistry andMolecular Biology (IUBMB)) include lipases within this group.

[0133] Examples include lipases selected from those classified under theEnzyme Classification (E.C.) numbers:

[0134] 3.1.1 (i.e. so-called Carboxylic Ester Hydrolases), including(3.1.1.3) Triacylglycerol lipases, (3.1.1.4.) Phosphorlipase A₂.

[0135] Examples of lipases include lipases derived from the followingmicroorganisms. The indicated patent publications are incorporatedherein by reference:

[0136] Humicola, e.g. H. brevispora, H. lanuginosa, H. brevis var.thermoidea and H. insolens (U.S. Pat. No. 4,810,414).

[0137] Pseudomonas, e.g. Ps. fragi, Ps. stutzeri, Ps. cepacia and Ps.fluorescens (WO 89/04361), or Ps. plantarii or Ps. gladioli (U.S. Pat.No. 4,950,417 (Solvay enzymes)) or Ps. alcaligenes and Ps.pseudoalcaligenes (EP 218 272) or Ps. mendocina (WO 88/09367; U.S. Pat.No. 5,389,536).

[0138] Fusarium, e.g. F. oxysporum (EP 130,064) or F. solani pisi (WO90/09446).

[0139] Mucor (also called Rhizomucor), e.g. M. miehei (EP 238,023).

[0140] Chromobacterium (especially C. viscosum).

[0141] Aspergillus (especially A. niger).

[0142] Candida, e.g. C. cylindracea (also called C. rugosa) or C.antarctica (WO 88/02775) or C. antarctica lipase A or B (WO 94/01541 andWO 89/02916).

[0143] Geotricum, e.g. G. candidum (Schimada et al., (1989), J.Biochem., 106, 383-388).

[0144] Penicillium, e.g. P. camembertii (Yamaguchi et al., (1991), Gene103, 61-67).

[0145] Rhizopus, e.g. R. delemar (Hass et al., (1991), Gene 109,107-113) or R. niveus (Kugimiya et al., (1992) Biosci. Biotech. Biochem56, 716-719) or R. oryzae.

[0146] Bacillus, e.g. B. subtilis (Dartois et al., (1993) Biochemica etBiophysica Acta 1131, 253-260) or B. stearothermophilus (JP 64/7744992)or B. pumilus (WO 91/16422).

[0147] Specific examples of readily available commercial lipases includeLipolase®, Lipolase™ Ultra, Lipozyme®, Palatase®, Novozym® 435,Lecitase® (all available from Novo Nordisk A/S).

[0148] Examples of other lipases are Lumafast™, Ps. mendocian lipasefrom Genencor Int. Inc.; Lipomax™, Ps. peudoalcaligenes lipase from GistBrocades/Genencor Int. Inc.; Fusarium solani lipase (cutinase) fromUnilever; Bacillus sp. lipase from Solvay enzymes. Other lipases areavailable from other companies.

[0149] Examples of lipase variants are described in e.g. WO 93/01285 andWO 95/22615.

[0150] The activity of the lipase can be determined as described in“Methods of Enzymatic Analysis”, Third Edition, 1984, Verlag Chemie,Weinhein, vol. 4, or as described in AF 95/5 GB (available on requestfrom Novo Nordisk A/S).

[0151] Oxidoreductases

[0152] Oxidoreductases (i.e. enzymes classified under the EnzymeClassification number E.C. 1 (Oxidoreductases) in accordance with theRecommendations (1992) of the International Union of Biochemistry andMolecular Biology (IUBMB)) include oxidoreductases within this group.

[0153] Examples include oxidoreductases selected from those classifiedunder the Enzyme Classification (E.C.) numbers:

[0154] Glycerol-3-phosphate dehydrogenase (NAD+) (1.1.1.8),Glycerol-3-phosphate dehydrogenase (NAD(P)⁺) (1.1.1.94),Glycerol-3-phosphate 1-dehydrogenase (NADP) (1.1.1.94), Glucose oxidase(1.1.3.4), Hexose oxidase (1.1.3.5), Catechol oxidase (1.1.3.14),Bilirubin oxidase (1.3.3.5), Alanine dehydrogenase (1.4.1.1), Glutamatedehydrogenase (1.4.1.2), Glutamate dehydrogenase (NAD(P)⁺) (1.4.1.3),Glutamate dehydrogenase (NADP⁺) (1.4.1.4), L-Amino acid dehydrogenase(1.4.1.5), Serine dehydrogenase (1.4.1.7), Valine dehydrogenase (NADP⁺)(1.4.1.8), Leucine dehydrogenase (1.4.1.9), Glycine dehydrogenase(1.4.1.10), L-Amino-acid oxidase (1.4.3.2.), D-Amino-acidoxidase(1.4.3.3), L-Glutamate oxidase (1.4.3.11), Protein-lysine6-oxidase (1.4.3.13), L-lysine oxidase (1.4.3.14), L-Aspartate oxidase(1.4.3.16), D-amino-acid dehydrogenase (1.4.99.1), Protein disulfidereductase (1.6.4.4), Thioredoxin reductase (1.6.4.5), Protein disulfidereductase (glutathione) (1.8.4.2), Laccase (1.10.3.2), Catalase(1.11.1.6), Peroxidase (1.11.1.7), Lipoxygenase (1.13.11.12), Superoxidedismutase (1.15.1.1).

[0155] Said glucose oxidases may be derived from Aspergillus niger.

[0156] Said laccases may be derived from Polyporus pinsitus,Myceliophtora thermophila, Coprinus cinereus, Rhizoctonia solani,Rhizoctonia praticola, Scytalidium thermophilum and Rhus vernicifera.

[0157] Bilirubin oxidases may be derived from Myrothechecium verrucaria.

[0158] The peroxidase may be derived from e.g. Soy bean, Horseradish orCoprinus cinereus.

[0159] The protein disulfide reductase may be any mentioned in any ofthe DK patent applications no. 768/93, 265/94 and 264/94 (Novo NordiskA/S), which are hereby incorporated as reference, including ProteinDisulfide reductases of bovine origin, Protein Disulfide reductasesderived from Aspergillus oryzae or Aspergillus niger, and DsbA or DsbCderived from Escherichia coli.

[0160] Specific examples of readily available commercial oxidoreductasesinclude Gluzyme™ (enzyme available from Novo Nordisk A/S). However,other oxidoreductases are available from others.

[0161] The activity of oxidoreductases and variants thereof can bedetermined as described in “Methods of Enzymatic Analysis”, thirdedition, 1984, Verlag Chemie, Weinheim, vol. 3.

[0162] Carbohydrases

[0163] Carbohydrases may be defined as all enzymes capable ofhydrolysing carbohydrate chains (e.g. starches) of especially five andsix member ring structures (i.e. enzymes classified under the EnzymeClassification number E.C. 3.2 (glycosidases) in accordance with theRecommendations (1992) of the International Union of Biochemistry andMolecular Biology (IUBMB)).

[0164] Examples include carbohydrases selected from those classifiedunder the Enzyme Classification (E.C.) numbers:

[0165] alpha-amylase (3.2.1.1) beta-amylase (3.2.1.2), glucanalpha-1,4glucosidase (3.2.1.3), cellulase (3.2.1.4),endo-beta-1,3(4)-glucanase (3.2.1.6), endo-beta-1,4-xylanase (3.2.1.8),dextranase (3.2.1.11), chitinase (3.2.1.14), polygalacturonase(3.2.1.15), lysozyme (3.2.1.17), beta-glucosidase (3.2.1.21),alpha-galactosidase (3.2.1.22), beta-galactosidase (3.2.1.23),amylo-1,6-glucosidase (3.2.1.33), xylan beta-1,4-xylosidase (3.2.1.37),glucan endo-beta-1,3-D-glucosidase (3.2.1.39), alpha-dextrinendo-1,6-glucosidase (3.2.1.41), sucrose alpha-glucosidase (3.2.1.48),glucan endo-1,3-alpha-glucosidase (3.2.1.59), glucanbeta-1,4-glucosidase (3.2.1.74), glucan endo-beta-1,6-glucosidase(3.2.1.75), arabinan endo-1,5-alpha-arabinosidase (3.2.1.99), lactase(3.2.1.108), chitonanase (3.2.1.132).

[0166] Examples of relevant carbohydrases include alpha-1,3-glucanasesderived from Trichoderma harzianum; alpha-1,6-glucanases derived from astrain of Paecilomyces; beta-glucanases derived from Bacillus subtilis;beta-glucanases derived from Humicola insolens; beta-glucanases derivedfrom Aspergillus niger; beta-glucanases derived from a strain ofTrichoderma; beta-glucanases derived from a strain of Oerskoviaxanthineolytica; exo-alpha-1,4-D-glucosidases (glucoamylases) derivedfrom Aspergillus niger; alpha-amylases derived from Bacillus subtilis;alpha-amylases derived from Bacillus amyloliquefaciens; alpha-amylasesderived from Bacillus stearothermophilus; alpha-amylases derived fromAspergillus oryzae; alpha-amylases derived from non-pathogenicmicroorganisms; alpha-galactosidases derived from Aspergillus niger;Pentosanases, xylanases, cellobiases, cellulases, hemi-cellulasesderiver from Humicola insolens; cellulases derived from Trichodermareesei; cellulases derived from non-pathogenic mold; pectinases,cellulases, arabinases, hemi-celluloses derived from Aspergillus niger;dextranases derived from Penicillium lilacinum; endoglucanase derivedfrom non-pathogenic mold; pullulanases derived from Bacillusacidopullyticus; beta-galactosidases derived from Kluyveromycesfragilis; xylanases derived from Trichoderma reesei.

[0167] Specific examples of readily available commercial carbohydrasesinclude Alpha-Gal™, Bio-Feed™ Alpha, Bio-Feed™ Beta, Bio-Feed™ Plus,Bio-Feed™ Plus, Novozyme® 188, Carezyme®, Celluclast®, Cellusoft®,Ceremyl®, Citrozym™, Denimax™, Dezyme™, Dextrozyme™, Finizym®,Fungamyl™, Gamanase™, Glucanex®, Lactozym®, Maltogenase™, Pentopan™,Pectinex™, Promozyme®, Pulpzyme™, Novamyl™, Termamyl®, AMG(Amyloglucosidase Novo), Maltogenase®, Sweetzyme®, Aquazym® (all enzymesavailable from Novo Nordisk A/S). Other carbohydrases are available fromother companies.

[0168] Lyases

[0169] Suitable lyases include Polysaccharide lyases: Pectate lyases(4.2.2.2) and pectin lyases (4.2.2.10), such as those from Bacilluslicheniformis disclosed in WO 99/27083.

[0170] Isomerases

[0171] Examples include isomerases selected from those classified underthe Enzyme Classification (E.C.) numbers (5.): e.g. xylose isomerase(5.3.1.5). An example of a relevant isomerase is the Protein DisulfideIsomerase, such as that described in WO 95/01425 (Novo Nordisk A/S). Aspecific example of a readily available commercial isomerase isSweetzyme®.

[0172] The activity of carbohydrases or variants thereof can bedetermined as described in “Methods of Enzymatic Analysis”, thirdedition, 1984, Verlag Chemie, Weinheim, vol. 4.

[0173] In another embodiment the test compound is any ingredient, suchas stabilizers used for the formulation of enzymes. In particular inrelation to the production of enzymes it is of interest to obtain enzymevariants having as little allergenicity as possible in order to reducethe risk of respiratory allergy of the people working in the enzymeindustry. Accordingly, when testing for test compounds, such as enzymesand variants thereof, suspected of causing respiratory allergy, it ispreferred to use a respiratory epithelial cell.

[0174] The test compound may be immunogenic itself or it may beconsidered as a hapten.

[0175] In one embodiment the test compound is any compound of a drugformulation, such as an active drug.

[0176] In another embodiment the test compound may be a compound used incosmetics.

[0177] In this case it is particularly contact allergy and a preferredfirst cell type is a keratinocyte.

[0178] Also, the test compound may be an organic solvent, a dye, or ametal.

[0179] According to the invention the test compound is added to the cellculture chosen for the purpose in a concentration sufficient to elicit adetectable cytokine response. The concentration of the test compound mayvary depending on the test compound in question. In one embodiment ofthe invention the test compound concentration may be 1, 10 or 100micrograms/ml. In another aspect the concentration of the test compoundadded to the cell culture may be dependent on the incubation time of thetest compound with the cell culture. In one embodiment of the inventionthe incubation time of the test compound may be from 0-16 hours, such as0, 2, 4, 6 or 16 hours.

[0180] In another aspect of the present invention the method accordingto the invention is used for assessing the toxicity of a test compound,comprising the step of:

[0181] (a) obtaining a predetermined and precharacterized cell culturecomprising at least one cell type of animal, including human, origincapable of substantially non-specific interaction with the testcompound, and of responding with a cytokine expression upon interaction,

[0182] (b) contacting the cell culture with the test compound,

[0183] (c) defining a specific cytokine profiles by determining for atleast one predetermined cytokine the cytokine response of the cellsexhibiting a substantially non-specific interaction with the testcompound, and

[0184] (d) correlating the cytokine profile to the toxicity of the testcompound.

[0185] As specified above with respect to the assessment of theallergenicity of a test compound it is possible to correlate thetoxicity of a test compound to the level of cytokines produced, in thatthe more toxic a compound the lower levels of cytokines are produced.Thus, the invention further relates to a method of simultaneousscreening of the toxicity of at least two test compounds, comprising thestep of:

[0186] (a) arranging a cell culture as defined above in at least twocompartments,

[0187] (b) contacting the cell cultures individually with a testcompound,

[0188] (c) defining specific cytokine profiles by determining thecytokine response of the respective cells exhibiting a substantiallynon-specific interaction with the test compound,

[0189] (d) correlating each cytokine profile to the toxicity of the testcompounds.

[0190] In yet a further aspect the invention relates to an assay kit forassessing the allergenicity or the toxicity for high through-putscreening of test compounds, wherein said assay kit comprises as partsof the kit:

[0191] (a) a cell culture comprising at least one animal, includinghuman, cell type,

[0192] (b) a cytokine determinant selected from at least one pair ofmonoclonal antibodies with specificity for a specific cytokine, at leastone cytokine-specific probe for mRNA detection, at least one set ofcytokine-specific primers for mRNA or cDNA detection,

[0193] (c) an assay device comprising at least two compartments.

[0194] In another embodiment of the invention the assay kit furtherrelates to at least one pair of monoclonal antibodies with specificityfor a specific membrane marker, at least one monoclonal antibody withspecificity for a specific membrane marker, at least one membranemarker-specific probe for mRNA detection, and at least one set ofmembrane marker-specific primers for mRNA or cDNA detection.

[0195] As described above the assay kit may comprise one cell type or acombination of cell types, the latter either co-cultured or culturedseparately. Furthermore, the assay kit may comprise medium for culturingthe cells. A person skilled in the art can easily define a mediumsuitable for culturing the cells specified herein.

[0196] Preferably the assay kit comprises an allergenicity standard,which is a standard for assessing the allergenicity of the testcompounds in question based on the cytokine profile determined. Theallergenicity standard may be in the form of information related to theassay kit, or it may be in the form of co-assessing the test compoundwith compounds of known allergenicity whereby the cytokine profilesobtained for the test compounds are correlated to the profiles obtainedfor known compounds.

[0197] The assay kit is preferably arranged for testing several testcompounds simultaneously, such as testing at least two compounds, morepreferably at least 10 test compounds simultaneously, and mostpreferably capable of assessing the allergenicity of about 100 testcompounds simultaneously.

[0198] The kit is to be considered as a kit in parts, that is the kitmay be combined from several different parts, such as a cell culturefrom one source, the determinant from another source, and the assaydevice from a third source.

[0199] The assay device is any suitable device, such as a plate with atleast one well, wherein the cells may be cultured and contacted with thetest compounds.

[0200] In a preferred embodiment the assay device comprises at least twocompartments in each well, whereby it is possible to co-culture twotypes of cells in the same well, and assess the cytokine response fromthe combination as described above. In one embodiment cell cultures maybe performed in 24, 48 or 96 well cell culture plates (Nunc). In orderto establish co-cultures, where cells are not allowed to have physicalcontact, specific inserts, figuring well defined permeable membranes,may be used (Nunc TC Inserts).

[0201] The assay kit may be used for assessing the allergenicity of anytest compound, as well as the toxicity of any test compound.

[0202] Experimentals

[0203] In the following the methods used in the examples are described.

[0204] Methods.

[0205] 1. Bioassay.

[0206] Human lung epithelial cells (BEAS-2B, ATCC # CRL-9609) wereinoculated in a culture flask (NUNC), precoated with 2% (vol/vol)Ultroser G in water, at 1500 to 3000 cells per cm², and were grown at37° C. in LHC-9 medium containing 2% (vol/vol) Ultroser G and 5% CO₂.

[0207] The cells were subcultured before reaching confluence. The mediumwas removed, and fresh 0.5% polyvinylpyrrolidone (PVP) in trypin (0.25%(wt/vol))-EDTA (0.03% (wt/vol)) solution was added until cells detached(usually after 5-10 minutes at room temperature). LHC-9 medium wasadded, and cells were collected by centrifugation (300× g for 15minutes), and resuspended in LHC-9 medium containing 2% (vol/vol)Ultroser G. Finally, cells were dispensed into precoated flasks (NUNC)at a density of 1500 to 3000 cells per cm^(2.)

[0208] When enough cells were available to perform the planned number ofassays, cells were trypinated, and were transferred to a 24-well cultureplate (NUNC) (1×10⁵ cells per well). Incubation was for 24 hours at 37°C. in LHC-9 medium containing 2% (vol/vol) Ultroser G and 5% CO₂. Then,the medium was removed and replaced by fresh prewarmed LHC-9 mediumcontaining 2% (vol/vol) Ultroser G and the protein under investigation.Protein-free medium was included as negative control, while cellsstimulated with 100 micrograms E. coli LPS 055:B5 were considered aspositive controls. Each assay was performed in triplicate.

[0209] The extracellular expression of selected cytokines was assessedby ELISA (R&D Systems), as a function of incubation time and proteinconcentration. ELISA was performed on the cell medium, directly aftercollection, or on medium that was stored at −20° C. or lower. Acalibration curve was included each time, to include quantification ofthe detected cytokine levels, as well as to assess assayreproducibility.

[0210] 2. Immunization of Brown Norway rats.

[0211] Twenty intratracheal immunizations were performed weekly with 100microliters 0.9% (wt/vol) NaCl (control group), or 100 microliters ofthe protein dilution mentioned before. Each group contained 10 rats.Blood samples (2 ml) were collected from the eye one week after everysecond immunization. Serum was obtained by blood clothing, andcentrifugation.

[0212] 3. IgE ELISA

[0213] The IgE ELISA was performed by using the following: Buffers andSolutions: Washing buffer PBS, 0.05% (v/v) Tween 20 Blocking buffer PBS,2% (wt/v) Skim Milk powder Dilution buffer PBS, 0.05% (v/v) Tween 20,0.5% (wt/v) Skim Milk powder Citrate buffer (0.1 M, pH 5.0-5.2)

[0214] Activation of CovaLink Plates:

[0215] Make a fresh stock solution of 10 mg cyanuric chloride per mlacetone.

[0216] Just before use, dilute the cyanuric chloride stock solution intoPBS, while stirring, to a final concentration of 1 mg/ml. Add 100microliters of the dilution to each well of the CovaLink NH₂ plates, andincubate for 5 minutes at room temperature. Wash 3 times with PBS.

[0217] Dry the freshly prepared activated plates at 50° C. for 30minutes. Immediately seal each plate with sealing tape. Preactivatedplates can be stored at room temperature for 3 weeks when kept in aplastic bag.

[0218] ELISA Procedure:

[0219] Mouse anti-Rat IgE was diluted 200×in PBS (5 micrograms/ml). 100microliters were added to each well. The plates were coated overnight at4° C.

[0220] Unspecific adsorption was blocked by incubating each well for 1hour at room temperature with 200 microliters blocking buffer. Theplates were washed 3×with 300 microliters washing buffer.

[0221] Unknown rat sera and a known rat IgE solution were diluted indilution buffer: Typically 10×, 20×and 40×for the unknown sera, and ½dilutions for the standard IgE starting from 1 microgram/ml. 100microliters were added to each well. Incubation was for 1 hour at roomtemperature.

[0222] Unbound material was removed by washing 3×with washing buffer.The anti-rat IgE (biotin) was diluted 2000×in dilution buffer. 100microliters were added to each well. Incubation was for 1 hour at roomtemperature. Unbound material was removed by washing 3×with washingbuffer.

[0223] Streptavidin was diluted 1000×in dilution buffer. 100 microliterswere added to each well. Incubation was for 1 hour at room temperature.Unbound material was removed by washing 3×with 300 microliters washingbuffer.

[0224] OPD (0.6 mg/ml) and H₂O₂ (0.4 microliter/ml) was dissolved incitrate buffer. 100 microliters were added to each well. Incubation wasfor 30 minutes at room temperature.

[0225] The reaction was stopped by adding 100 microliters H₂SO₄. Theplates were read at 492 nm with 620 nm as reference.

EXAMPLE 1

[0226] Identification of Cytokines that can be Induced in EpithelialCells

[0227] In this set of experiments, 100 micrograms LPS(lipo-polysaccharide) were used to stimulate the cells. ELISA wasperformed after 2, 4, 6, 8 and 16 hours. Table 2 shows the cytokinesinduced in the cells (+), and does also indicate the cytokines notdetected in the epithelial cell medium when cells are stimulated withLPS (−) TABLE 2 Cytokine Cell response IL-1α − IL-1β − IL-2 − IL-4 −IL-5 − IL-6 + IL-8 + IFN-γ − TNF-α − GM-CSF + MCP-1 + MIP-1β − RANTES +

[0228] The FIGS. 1A-D show the different kinetics for the cytokinesindicated in the table to be positive (+). As IL-8 and IL-6 revealedsimilar kinetics, only IL-8 is shown (FIG. 1).

EXAMPLE 2

[0229] Assessment of the Cytokine Levels Induced in Epithelial Cells byVarious Protease Derivatives

[0230] Protease P from the subtilisin family (CDJ31: A Bacilluslicheniformis serine protease (E.C. 3.4.21.62)) was selected, andmodified with polyethylen-glycol (PEG) molecules of different size,specifically 350, 750, 1000, 2000 and 5000 Da. Both unmodified andmodified enzymes were assessed in the epithelial celll assay for therepotency to induce extracellular cytokine production.

[0231] To prevent proteolysis of the cytokines by the protease, thelatter was inhibited by PMSF (10 mM), prior to the dilution in the cellmedium. Typically, PMSF was diluted about 1000×before being applied onthe cells. At these concentrations, PMSF and its hydrolysis products hadno detectable effect on cytokine production.

[0232]FIG. 2 shows the typically IL-8, IL-6 and MCP-1 kinetics forunmodified and modified protease. The latter is represented byP-bis-S-PEG2000. The baseline is obtained with reference to the controlmedium. Almost identical results were observed for another subtilisinprotease (PD498: A Bacillus sp. Serine protease (E.C. 3.4.21.66)).

[0233] For comparison of the respective cytokine levels, the levelsresulting after 4 hours of incubation with 100 micrograms of proteinwere selected.

EXAMPLE 3

[0234] Correlation of the Detected Cytokine Levels and IgE Levels inRats with the Length of the PEG-Molecules Used for Enzyme Modification

[0235] Rats were immunized intratracheally with unmodified and modifiedprotease, and the protease specific IgE levels were detected by ELISA.The IgE levels detected throughout the study were integrated, andcompared relative to the levels observed in rats immunized withunmodified enzyme. In FIG. 3, (a) shows a decrease in relative IgElevels with increasing length of the PEG molecules used to modified theenzyme. IL-8 levels (b) were found to increase with increasing PEG size,while both IL-6 (c) and MCP-1 (d) levels revealed bell-shaped kinetics.

EXAMPLE 4

[0236] Correlation of the Detected Cytokine Levels with IgE Levels inRats as Compared to the Levels Detected for the Unmodified Enzyme

[0237] Using the results obtained in Example 3 it is possible directlyto correlate the dettected cytokine levels with IgE levels in rats.

[0238] In FIG. 4, (a) shows a decrease in IL-8 levels with increasingIgE levels as compared to the unmodified enzyme. IL-6 (b) and MCP-1 (c)IgE levels steeply increased followed by a steep decline in IgE levelsas compared to the unmodified enzyme.

[0239] The correlation between IgE and the respective cytokines can besummarized as follows: TABLE 3 IgE levels in rats (%) IL-8 IL-6MCP-1 >50 − − − 50-25 + + −(p > 0.05) 25-10 + + + <10 + − +

EXAMPLE 5

[0240] Comparison Between the Detected Cytokine Levels and IgE Levels inRats of a Protease and Lipolase

[0241] The cytokine levels of serine protease E.C. 3.4.21.66 fromBacillus sp. and triacylglycerol acylhydrolase (lipase) E.C. 3.1.1.3from Humicola lanuginosa were assessed in the epithelial cell assay.Rats were immunized intratracheally with the same protease and lipase,and the IgE levels were detected by ELISA. FIG. 5 shows the normalizedcytokine levels as well as IgE levels of both the protease and thelipase.

[0242] Based upon the results shown in table 3 of example 3, theprotease would be more allergenic than the lipase. Accordingly, the lowlevel of all cytokines observed for the protease corresponded to a highIgE level, while the high level of all cytokines observed for the lipasecorresponded to a significantly lower IgE level.

1. A method for the in vitro assessment of the allergenicity of a testcompound, comprising the step of: (a) obtaining a predetermined andprecharacterized cell culture comprising at least one cell type ofanimal, including human, origin capable of substantially non-specificinteraction with the test compound, and of responding with a change incytokine expression upon interaction, (b) contacting the cell culturewith the test compound, (c) defining a specific cytokine profile bydetermining for at least one predetermined cytokine the cytokineresponse of the cells exhibiting a substantially non-specificinteraction with the test compound, and (d) correlating the cytokineprofile to the allergenicity of the test compound.
 2. A method for thesimultaneous screening of the allergenicity of at least two testcompounds, comprising the step of: (a) arranging a specific cell type inat least two separate compartments of a cell culture of claim 1, (b)contacting the separated cell culture compartments individually with atest compound, (c) defining specific cytokine profiles by determiningthe cytokine responses of the respective cells exhibiting asubstantially non-specific interaction with the test compound, (d)correlating each cytokine profile to the allergenicity of the testcompounds.
 3. The method of claim 1 or 2, wherein the at least one celltype is epithelial cells.
 4. The method of claim 3, wherein theepithelial cells are respiratory tract epithelial cells.
 5. The methodof claim 3, wherein the epithelial cells are gastro-intestinal tractepithelial cells.
 6. The method of claim 1 or 2, wherein the at leastone cell type is keratinocytes.
 7. The method of claim 1 or 2, whereinthe at least one cell type is dendritic cells.
 8. The method of claim 1or 2, wherein the at least one cell type is macrophages.
 9. The methodof claim 1 or 2, wherein the at least one cell type is mast cells. 10.The method of claim 1 or 2, wherein the at least one cell type ismonocytes.
 11. The method of claim 1 or 2, wherein the at least one celltype is endothelial cells.
 12. The method of any of the precedingclaims, comprising the step of: (a) obtaining at least two cellcultures, each comprising one cell type, (b) contacting each cellculture with the test compound, (c) defining specific cytokine profilesby determining the cytokine responses of the respective cell cultures,each exhibiting a substantially non-specific interaction with the testcompound, (d) correlating the cytokine profile to the allergenicity ofthe test compound.
 13. The method of claim 12, wherein the two celltypes are co-cultured.
 14. The method of claim 12, wherein the cytokineresponses of the two cell types are determined for each of theindividual cell types.
 15. The method of claim 12, wherein the cytokineresponses of the two cell types are determined as one response.
 16. Themethod of claim 12, wherein the two cell types of the co-culture arephysically separated.
 17. The method of any of claims 12-16, wherein onecell type is epithelial cells and the other is dendritic cells.
 18. Themethod of any of claims 12-16, wherein one cell type is epithelial cellsand the other is macrophages.
 19. The method of any of claims 12-16,wherein one cell type is epithelial cells and the other is mast cells.20. The method of any of claims 12-16, wherein one cell type isepithelial cells and the other is monocytes.
 21. The method of any ofclaims 12-16, wherein one cell type is epithelial cells and the other isendothelial cells.
 22. A method for the in vitro assessment of thetoxicity of a test compound, comprising the step of: (a) obtaining apredetermined and precharacterized cell culture comprising at least onecell type of animal, including human, origin capable of substantiallynon-specific interaction with the test compound, and of responding witha cytokine expression upon interaction, (b) contacting the cell culturewith the test compound, (c) defining a specific cytokine profile bydetermining for at least one predetermined cytokine the cytokineresponse of the cells exhibiting a substantially non-specificinteraction with the test compound, and (d) correlating the cytokineprofile to the toxicity of the test compound.
 23. A method for thesimultaneous screening of the toxicity of at least two test compounds,comprising the step of: (a) arranging a cell culture of claim 22 in atleast two compartments, (b) contacting the cell cultures individuallywith a test compound, (c) defining specific cytokine profiles bydetermining the cytokine responses of the respective cells exhibiting asubstantially non-specific interaction with the test compound, (d)correlating each cytokine profile to the toxicity of the test compounds.24. The method of any of claims 1-23, wherein the test compound is aprotein.
 25. The method of any of claims 1-23, wherein the test compoundis a glycoprotein.
 26. The method of any of claims 1-23, wherein thetest compound is a lipoprotein.
 27. The method of any of claims 1-23,wherein the test compound is a proteolipid.
 28. The method of any ofclaims 1-23, wherein the test compound is a phospholipid.
 29. The methodof any of claims 24-28, wherein the test compound is an enzyme or anenzyme variant.
 30. The method of any of claims 1-29, wherein the testcompound is for the formulation of enzymes.
 31. The method of any ofclaims 1-21, wherein the test compound is a hapten.
 32. The method ofany of claims 1-31, wherein the test compound is a drug.
 33. The methodof claim 31, wherein the test compound is a cosmetic compound.
 34. Themethod of claim 22 or 23, wherein the test compound is an organicsolvent.
 35. The method of claim 31, wherein the test compound is a dye.36. The method of claim 31, wherein the test compound is a metal. 37.The method of any of the preceding claims, wherein the at least one celltype is derived from human tissue.
 38. The method of any of claims 1-37,wherein the at least one cell type is derived from human blood.
 39. Themethod of any of the preceding claims, wherein the extracellularcytokine response is determined by analysis with enzyme-linkedimmunosorbent assay.
 40. The method of any of claims 1-38, wherein theintracellular cytokine response is determined by analysis withenzyme-linked immunosorbent assay.
 41. The method of any of claims 1-38,wherein the cytokine response is determined by in-situ-hybridizationtechnique.
 42. The method of any of claims 1-38, wherein the cytokineresponse is determined by analysis with polymerase-chain-reaction (PCR)technique.
 43. The method of claim 42, wherein the cytokine response isdetermined by analysis with quantitative-PCR technique.
 44. The methodof claim 42, wherein the cytokine response is determined by analysiswith in-situ-PCR technique.
 45. The method of any of claims 41-44,wherein intracellular mRNA is determined.
 46. The method of claim 1 or22, wherein membrane markers induced by the test compound in cellsexhibiting a substantially non-specific interaction with the testcompound, are determined.
 47. The method of claim 46, wherein themembrane markers assayed for are VCAM-1 or ICAM-1.
 48. The method of anyof the preceding claims, wherein more than one cytokine is being assayedfor.
 49. The method of claim 48, wherein at least two cytokines arebeing assayed for.
 50. The method of claim 48, wherein at least fourcytokines are being assayed for.
 51. The method of claim 48, wherein thecytokines assayed for are interleukin-8, interleukin-6, MCP-1, andGM-CSF.
 52. The method of claim 1, wherein the level and number ofcytokines assessed are factors correlating to the allergenic potency ofthe test compound.
 53. The method of claim 52, wherein the level andnumber of cytokines assessed are factors correlating to low allergenictest compounds.
 54. The method of claim 52, wherein the level and numberof cytokines assessed are factors correlating to high allergenic testcompounds.
 55. The method of claim 23, wherein the level and number ofcytokines assessed are factors correlating to the toxic potency of thetest compound.
 56. The method of claim 55, wherein the level and numberof cytokines assessed are factors correlating to low toxic testcompounds.
 57. The method of claim 55, wherein the level and number ofcytokines assessed are factors correlating to high toxic test compounds.58. A method of characterizing a cytokine response of a cell type asdescribed in any of the preceding claims, comprising the step of: (a)obtaining a predetermined cell culture comprising one cell type ofanimal, including human, origin capable of substantially non-specificinteraction with the test compound, and of responding with a cytokineexpression upon interaction, (b) identifying the cytokines that can beexpressed by the cell culture upon non-specific interaction with thetest compound by using a non-specific multivalent inducer, (c)contacting a part of the cell culture with a test compound, determiningthe level of the identified cytokines obtaining a cytokine profile, (d)immunizing an animal with the test compound and determining theresulting IgE level of the animal, (e) correlating the cytokine profileto the IgE level determined, and (f) repeating step c)-e) until at leasta test compound of a high IgE level, a test compound of a low IgE leveland a test compound of a medium IgE level have been tested.
 59. An assaykit for high through-put screening of the allergenicity or toxicity of atest compound, comprising: (a) a cell culture comprising at least oneanimal, including human, cell type, (b) a cytokine determinant selectedfrom at least one monoclonal antibody with specificity for a specificcytokine, at least one cytokine-specific probe for mRNA detection, atleast one set of cytokine-specific primers for mRNA or cDNA detection,(c) an assay device comprising at least two compartments.
 60. The assaykit as described in claim 59, comprising at least one monoclonalantibody with specificity for a specific membrane marker, at least onemonoclonal antibody with specificity for a specific membrane marker, atleast one membrane marker-specific probe for mRNA detection, and atleast one set of membrane marker-specific primers for mRNA or cDNAdetection.
 61. The assay kit of claim 59 or 60, further comprising anallergenicity standard.
 62. The use of an assay kit of any of claims59-61 for the high through-put screening of the allergenicity of atleast two test compounds.
 63. The use of an assay kit of claim 62 forassessing the allergenicity of at least 10 test compoundssimultaneously.
 64. The use of claim 62 for assessing the allergenicityof about 100 test compounds simultaneously.
 65. The use of an assay kitof claim 60 for the high through-put screening of the toxicity of atleast two test compounds.
 66. The use of an assay kit of claim 65 forassessing the toxicicity of at least 10 test compounds simultaneously.67. The use of claim 65 for assessing the toxicity of about 100 testcompounds simultaneously.